There are two major hurdles that must be overcome before cancer gene therapy becomes a clinical reality: tumor-specific therapeutic gene activation and efficient delivery of gene therapy vectors into the tumor mass. The ability to specifically activate therapeutic genes in cancer cells is what differentiates gene therapy from conventional cancer therapies. In the previous funding cycle, we have made substantial progress in developing a novel gene therapy approach by which therapeutic genes can be regulated by hyperthermia. We have shown that therapeutic genes can be regulated by hyperthermia in a very efficient and targeted fashion in the tumor mass with impressive anti-tumor efficacy in experimental tumor models. However, we are still faced with the problem of inefficient delivery of the gene therapy vectors, which can serious hinder the application of our otherwise very promising strategy. In this application, we want to build upon our previous successes and continue to improve our hyperthermia-regulated gene therapy approach. We will deal with both the regulation issue and the delivery issue for gene therapy. A two-pronged approach will be employed. On the one hand, we will continue to explore new gene regulation approaches that may further improve/enhance our heat-induced therapeutic gene activation strategy. On the other hand, we will tackle the gene delivery issue by developing hyperthermia-regulated replication competent adenovirus vectors that can selectively replicate in the tumor mass. In particular, we will have two specific aims. In specific aim 1, we will develop a novel gene regulation strategy that may further enhance hyperthermia-activated therapeutic gene expression. For this specific aim, we will attempt to design a novel Cre-lox based irreversible genetic switch that can potentially enhance the regulation and expression of heat-activated gene therapy. In specific aim 2, we will develop conditionally replicative adenovirus vectors with virus replication under the control of hyperthermia or hypoxia. We will engineer recombinant adenovirus vectors that can selectively replicate in hyperthermia-treated tumor cells or those that can selectively replicate in hypoxic tumor cells. The well-known anti-angiogenic gene endostatin will be engineered into the vectors. The vectors will then be evaluated for their anti-tumor efficacy either alone or in combination with hyperthermia and/or ionizing radiation. At the end of the new funding cycle, we hope to make significant advancement in both the delivery and the regulation of the hyperthermia-mediated gene therapy approach, thereby making it even closer to clinical human cancer treatment.